Abstract
Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.
Highlights
UV irradiation induces various lesions in DNA [1]
The present study using FAD-induced paramagnetic relaxation enhancement (PRE) shows that the distance between FAD and cyclobutane pyrimidine dimer (CPD) in the T. thermophilus CPD photolyase-DNA complex is ϳ16 Å
The intensity reduction ratios and the distances fit well with the Solomon-Bloembergen equation (Fig. 2). These results suggest that the intensity reduction observed in the radical semiquinone form was derived predominantly from FAD-induced PRE
Summary
UV irradiation induces various lesions in DNA [1]. The cyclobutane pyrimidine dimer (CPD) is a major DNA lesion induced by UV light [2]. The crystal structures of the CPD photolyases from Escherichia coli, Anacystis nidulans, and Thermus thermophilus HB8 were recently solved (8 –10). These structures show a similar global fold, and they have an FAD-containing cavity in the C-terminal helical domain. The crystal structure of the T. thermophilus CPD photolyase-thymine complex has been solved [10] In this structure, the thymine molecule resided in the cavity, suggesting that this cavity forms the CPD-binding site. The structure of a CPD photolyase in complex with CPD-containing DNA has not yet been determined. PRE would be useful for determining the distance between FAD and CPD in the CPD photolyase-DNA complex, because the FAD in CPD photolyase can assume the radical semiquinone form [4]
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